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At least 721 records · Page 40Linked to original sources
PubMed1997

Early events leading to fate decisions during leech embryogenesis.

This paper reviews leech development up to the 12-cell embryo. Oogenesis proceeds by a system of nurse cells that contribute to oocyte growth via continuous cytoplasmic connections. Development begins when fertilized eggs are deposited: formation of the polar bodies, and centration of the male and female pro-nuclei is accompanied by cytoskeletal contractions, and formation of teloplasm (yolk-free cytoplasm). The first cleavages are asymmetric: cell D', the largest macromere in the eight-cell embryo, contains most of the teloplasm. At fourth cleavage D' divides equally; its animal and vegetal daughters are precursors of segmental ectoderm and mesoderm, respectively. Teloplasm is a determinant of the D' cell fate. The expression pattern of Hro-nos, a leech homolog to the Drosophila gene nanos, suggests that it may be a determinant associated with the animal cortex and inducing the ectodermal fate in the animal daughter cell of the D' macromere.

Journal Article↗
PubMed2005

Sodium-dependent potassium channels in leech P neurons.

In leech P neurons the inhibition of the Na(+)-K(+) pump by ouabain or omission of bath K(+) leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K(+) and Na(+) are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K(+) conductance of the plasma membrane, which compensates for the reduction of the K(+) gradient. The data presented here strongly suggest that the increased K(+) conductance is due to Na(+)-activated K(+) (K(Na)) channels. Specifically, an increase in the cytosolic Na(+) concentration ([Na(+)](i)) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R(in)) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R(in) and [Na(+)](i) followed a simple model in which the R(in) decrease was attributed to K(+) channels that are activated by the binding of three Na(+) ions, with half-maximal activation at [Na(+)](i) between 45 and 70 mM. At maximum channel activation, R(in) was reduced by more than 90%, suggesting a significant contribution of the K(Na) channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K(Na) channels in leech P neurons are also activated by Li(+).

Animals↗
PubMed2005

Mechanisms of postinhibitory rebound and its modulation by serotonin in excitatory swim motor neurons of the medicinal leech.

Postinhibitory rebound (PIR) is defined as membrane depolarization occurring at the offset of a hyperpolarizing stimulus and is one of several intrinsic properties that may promote rhythmic electrical activity. PIR can be produced by several mechanisms including hyperpolarization-activated cation current (I(h)) or de-inactivation of depolarization-activated inward currents. Excitatory swim motor neurons in the leech exhibit PIR in response to injected current pulses or inhibitory synaptic input. Serotonin, a potent modulator of leech swimming behavior, increases the peak amplitude of PIR and decreases its duration, effects consistent with supporting rhythmic activity. In this study, we performed current clamp experiments on dorsal excitatory cell 3 (DE-3) and ventral excitatory cell 4 (VE-4). We found a significant difference in the shape of PIR responses expressed by these two cell types in normal saline, with DE-3 exhibiting a larger prolonged component. Exposing motor neurons to serotonin eliminated this difference. Cs+ had no effect on PIR, suggesting that I(h) plays no role. PIR was suppressed completely when low Na+ solution was combined with Ca2+-channel blockers. Our data support the hypothesis that PIR in swim motor neurons is produced by a combination of low-threshold Na+ and Ca2+ currents that begin to activate near -60 mV.

Animals↗
PubMed2005

Three-dimensional culture of leech and snail ganglia for studies of neural repair.

Three-dimensional (3D) collagen gels provide a stable matrix in which isolated regenerating ganglia from leech and snail can be maintained for studies of the molecular and cellular mechanisms underlying the regenerative process. Segmental ganglia from leech, or supraoesophageal, suboesophageal or buccal ganglia from snail were maintained for up to 3 weeks in 3D matrices of mammalian Type I collagen. The collagen matrix supports the regenerative outgrowth of axon tracts as well as the migration of microglial cells, important elements in the repair process. Proteins or soluble factors or target tissue may be added to the basic collagen matrix to manipulate the environment of the regenerating tissue. We describe techniques for immunostaining of regenerating axons and microglial cells within the gel matrix in combination with staining of cell nuclei, and the use of intracellular labelling to distinguish axons of identified neurons within the regenerative outgrowth.

Animals↗
PubMed1987

Rapid purification and revised amino-terminal sequence of hirudin: a specific thrombin inhibitor of the bloodsucking leech.

Hirudin is a specific polypeptide thrombin inhibitor consisting of 65 amino acids that is produced by the leech, Hirudo medicinalis. We describe a rapid method for the purification of hirudin from a leech extract. Crude hirudin, purchased from a commercial source, was first fractionated on a DEAE-HPLC column using a salt gradient. Hirudin activity was monitored by inhibition of the thrombin-mediated hydrolysis of a synthetic substrate H-D-Phenylalanyl-Pipecolyl-Arginine-p-Nitroanilide. The fractions containing antithrombin activity were pooled and further purified by reverse-phase HPLC. The homogeneity of purified hirudin was confirmed by a single amino-terminal sequence for 43 residues with Val-Val as the first two amino acids. Residue 33 was Asn rather than Asp as reported previously.

Amino Acid Sequence↗
PubMed1989

The "chemical leech": intra-replant subcutaneous heparin as an alternative to venous anastomosis. Report of three cases.

Three successful cases of distal finger replantation are described where suitable veins were unavailable for anastomosis after arterial flow had been re-established. To prevent infarction, calcium heparin was injected subcutaneously into the replants at intervals over 9 days. This allowed the venous blood to escape into the dressings while an adequate microvenous circulation was re-established, thus simulating the effect of a leech. Complete survival of all three distal replantations was observed. No attempted case has been unsuccessful. The procedure is simple and can be administered by the nursing staff. It avoids some of the problems associated with the use of medicinal leeches and systemic heparin. Applications of this technique in other areas of replantation and flap surgery are suggested.

Adult↗
PubMed2006

Acetyl-L-carnitine affects nonassociative learning processes in the leech Hirudo medicinalis.

Acetyl-L-carnitine is a natural molecule widely distributed in vertebrate and invertebrate nervous system. It is known to have significant effects on neuronal activity playing a role as neuroprotective and anti-nociceptive agent, as well as neuromodulatory factor. About its capability of affecting learning processes the available data are controversial. In the present study, we utilized the simplified model system of the leech Hirudo medicinalis to analyze the effects of acetyl-L-carnitine, assessing whether and how it might affect elementary forms of nonassociative learning processes. In leeches with the head ganglion disconnected from the first segmental ganglion, repetitive application of weak electrical shocks onto the caudal portion of the body wall induces habituation of swim induction whereas brush strokes on the dorsal skin produces sensitization or dishabituation when the nociceptive stimulus is delivered on previously habituated animals. Herein, the effects of different concentrations of acetyl-L-carnitine (2 mM - 0.05 mM) have been tested at different times on both sensitization and dishabituation. The results show that a single treatment of acetyl-L-carnitine blocked the onset of sensitization in a dose- and time-dependent manner. In fact, the most effective concentration able to block this process was 2 mM, which induced its major effects 11 days after the treatment, whereas 0.05 mM was unable to affect the sensitization process at all considered time points. On the contrary, acetyl-L-carnitine did not completely abolish dishabituation at the tested concentrations and at every time point. Finally, acetyl-L-carnitine also impaired the habituation of swim induction, but only 11 days after treatment.

Acetylcarnitine↗
PubMed2005

Functional screening of serine protease inhibitors in the medical leech Hirudo medicinalis monitored by intensity fading MALDI-TOF MS.

The blood-feeding invertebrates are a rich biological source of drugs and lead compounds to treat cardiovascular diseases because they have evolved highly efficient mechanisms to feed on their hosts by blocking blood coagulation. In this work, we focused our attention on the leech Hirudo medicinalis. We performed, by "intensity fading" MALDI-TOF mass spectrometry, a comprehensive detection and functional analysis of pre-existent peptides and small proteins with the capability of binding to trypsin-like proteases related to blood coagulation. Combining "intensity fading MS" and off-line LC prefractionation allowed us to detect more than 75 molecules present in the leech extract that interact specifically with a trypsin-like protease over a sample profile of nearly 2,000 different peptides/proteins in the 2-20-kDa range. Moreover we resolved 232 individual components from the complex mixture, 13 of which have high sequence homology with previously described serine protease inhibitors. Our findings indicate that such extracts are much more complex than expected. Additionally, intensity fading MS, when complemented with LC separation strategies, seems to be a useful tool to investigate complex biological samples, establishing a new bridge between profiling, functional peptidomics, and subsequent drug discovery.

Amino Acid Sequence↗
PubMed1986

Cloning and expression in Escherichia coli of a synthetic DNA for hirudin, the blood coagulation inhibitor in the leech.

A 235-bp DNA coding for the leech blood coagulation inhibitor, hirudin, was chemically synthesized. The synthesis involved preparation of seven long oligodeoxyribonucleotide pairs which were assembled and cloned using a rapid and simple procedure. More than half of the transformed Escherichia coli cells expressed a biosynthetic polypeptide having biological properties which were very similar to authentic hirudin from the leech Hirudo medicinalis. To achieve efficient expression, we fused the hirudin DNA to a truncated C1 repressor gene of bacteriophage lambda to create a hybrid protein. An additional methionine at the fusion point allowed the active hirudin to be cleaved off by cyanogen bromide.

Animals↗
PubMed1986

Cleft lip surgery in Anglo-Saxon Britain: the Leech Book (circa A.D. 920).

The Leech Book, the oldest known Anglo-Saxon herbarium, probably written in Winchester, circa A.D. 920, by Cyril Bald or at his special request, contains a short chapter on the surgical treatment of the cleft lip; this chapter apparently represents the first record in a medical manuscript of this treatment. The original Anglo-Saxon text is presented together with transcriptions into more modern English. The general value of the Leech Book is briefly studied.

Cleft Lip↗
PubMed2000

Linear and nonlinear measures predict swimming in the leech

Stimulation of a trigger interneuron of an isolated nerve cord preparation of the medicinal leech, Hirudo medicinalis, sometimes leads to swimming; sometimes it does not. We investigate signals transmitted in the ventral cord of the leech after stimulation and seek quantitative measures that would make it possible to distinguish signals that predict swimming from those that do not. We find that a number of linear as well as nonlinear measures provide statistically significant distinctions between the two kinds of signals. The linear measures are the time dependence of (i) the standard deviation and (ii) the autocorrelation function at a small time delay. The nonlinear measures are (i) a measure of nonlinear predictability and (ii) the time dependence of a measure of the size of the embedded signal trajectory. Calculations using surrogate data suggest that the differences between the two classes of signals are dynamical as well as statistical.

Journal Article↗
PubMed1993

Novel hirudin variants from the leech Hirudinaria manillensis. Amino acid sequence, cDNA cloning and genomic organization.

Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion-exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2) and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C-terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the C-terminal portion of the molecule which is replaced by aspartic acid. Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin-like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported. The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides. HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.

Amino Acid Sequence↗
PubMed1970

Physiological and morphological properties of motoneurones in the central nervous system of the leech.

1. A number of motor cell bodies have been identified in the segmental ganglia of the ventral nerve cord of the medicinal leech. These motoneurones supply either excitatory or inhibitory innervation to the muscles in the body wall.2. Several tests were made to establish that each of the identified motoneurones directly innervates muscle fibres. (a) By injecting a fluorescent dye into the cell bodies of motoneurones, their axons were traced through one or both contralateral roots. (b) Electrical stimulation of a motoneurone by an intracellular electrode caused a single nerve impulse to travel through the roots to the muscles where it set up an excitatory or an inhibitory junctional potential. (c) Impulses set up in the roots were conducted antidromically to the cell body. (d) If the preparation was bathed in 20 mM-Mg(2+), which blocks chemical synapses, conduction from the cell body to the muscles was not interrupted. Thus it is unlikely that an interneurone was interposed in the pathway within the ganglion.3. Fourteen pairs of excitatory cells and three pairs of inhibitory cells can be identified in each of the twenty-one segmental ganglia. These neurones together supply the five different muscle layers in each segment which execute the movements of the leech. Each neurone innervates a territory of muscle fibres which has a consistent size and location from segment to segment. Several lines of evidence suggest that the identified cells form a major fraction of the total number of excitatory motoneurones in the ganglion.4. The territories of the motoneurones are arranged in a quilt-like pattern closely resembling that already found for the receptive fields of sensory cells on the skin. Within the longitudinal muscle sheet, individual cells supply narrow or wide strips. The firing of each cell thus could produce bending of the segment in various directions or symmetrical shortening of it, depending on which of the motoneurones were active.5. It is possible to deduce which motoneurones are firing to produce a particular movement of the animal. Thus these experiments provide a basis for studying reflex integration between motoneurones and the identified mechanosensory cells in the ganglion.

Animals↗
PubMedDate not supplied

[Redistribution of membrane-bound Ca2+ in neurolemma of leech Retzius-neurons during thermostimulation].

Changes in electrical activity, plasma membrane and inner mitochondrial membrane potentials, as well as in the content of membrane-bound calcium and NADH in identified leech ganglion neurons during thermal stimulation were studied by microelectrode and microfluorimetric methods. It was shown that thermal stimulation of skin patches from different regions of leech body causes a reversible increase in the firing rate of pacemaker neurons (Rz neurons). A decrease in the content of membrane-bound calcium in the neurolemma and NADH content in Rz neuron mitochondria, and a depolarization of inner mitochondrial membrane were also observed.

Cell Membrane↗
PubMed1998

Long-lasting depolarization of leech neurons mediated by receptors with a nicotinic binding site

The serotonergic Retzius neurons of the leech midbody ganglia respond in a complex manner to pressure pulses of acetylcholine (ACh) applied onto their soma with a fast depolarization followed by a slower hyperpolarization and an additional delayed long-lasting depolarization. The delayed depolarization is the subject of the present study. The delayed depolarization could be elicited by long (>1 s) ACh pressure pulses or by short pulses (10 ms) of carbachol, nicotine and DMPP, but not by muscarinic agonists. It was inhibited by bath application of nicotine (10-100 micromol l-1), strychnine (100 micromol l-1) and atropine (10-100 micromol l-1). Nicotinic antagonists that blocked the fast depolarization and the slow hyperpolarization (100 micromol l-1 mecamylamine and d-tubocurarine) did not affect the delayed depolarization induced by carbachol. Partial replacement of the extracellular Na+ by glucamine caused a decrease in the amplitude of the response and a shift of its reversal potential to more negative values. Carbachol pulses applied to Retzius neurons of the ganglia innervating the reproductive segments elicited delayed depolarizations of much smaller amplitude than the ones recorded in Retzius neurons from standard segments. The delayed depolarization could be elicited by the application of short agonist pulses onto different loci over the surface of the ganglion, at a distance from the soma. Isolated cultured Retzius neurons did not exhibit the delayed depolarization although they readily expressed the earlier phases of the complex cholinergic response. Carbachol pulses applied to the soma of other neurons in the leech ganglion produced a variety of specific responses.The results suggest that the delayed depolarization was produced by the activation of a cationic conductance mediated by receptors with a pharmacological profile similar to that of the 9 nicotinic receptors and was not a byproduct of the early phases of the cholinergic response. The response seemed to be initiated in the extensive neuropilar processes of the Retzius cell, enabling a persistent excitatory signal.

Journal Article↗
PubMed1996

Isolation, characterization, and expression of Le-msx, a maternally expressed member of the msx gene family from the glossiphoniid leech, Helobdella.

The msx gene family is one of the most highly conserved of the nonclustered homeobox-containing genes. We have isolated an msx homolog (Le-msx) from the glossiphoniid leech, Helobdella robusta, and characterized its pattern of expression by whole mount in situ hybridization. In situ expression and reverse transcription polymerase chain reaction (RT-PCR) data results show that Le-msx is a maternal transcript initially uniformly distributed in the cortex of immature oocytes that becomes asymmetrically localized to the polar regions of the uncleaved zygote. This is the earliest reported expression for the msx gene family and the first maternally expressed homeodomain-containing transcription factor reported in annelids. During embryonic development, Le-msx is expressed in all 10 embryonic stem cells and their segmental founder cell descendants. At midembryonic stages, Le-msx is expressed in the expanding germinal plate. Le-msx is confined to the central nervous system and nephridia at late (stage 9) stages and subsequently disappears from nephridia. In addition, we present a phylogenetic hypothesis for the evolution of the msx gene family, including the identification of a putative C. elegans msx homolog and the realignment of the sponge msx homolog to the NK class of homeodomain genes.

Amino Acid Sequence↗
PubMed1996

Segmental diversification of an identified leech neuron correlates with the segmental domain in which it expresses Lox2, a member of the Hox gene family.

The cellular colocalization of LOX2 protein and small cardioactive peptide (SCP)-like immunoreactivity was studied in the nerve cord of the glossiphoniid leech Helobdella triserialis. Of the six neurons that express SCP in the midbody segments 7 to 17, only one, the MPS neuron, expresses LOX2 protein. The medial paired SCP (MPS) neurons are segmentally repeated and can be divided into three contiguous segmental domains according to cell body size and the timing and level of SCP expression. MPS neurons located in the anterior and middle segmental domains express LOX2 protein. In the middle domain, large MPS neurons begin to accumulate SCP shortly after the end of embryonic development, whereas in the anterior domain the MPS neurons are smaller and begin to express SCP at a later stage. In the posterior domain the MPS neurons exhibit a third phenotype -- they have large cell bodies, express low levels of SCP starting from the midjuvenile stage, and do not show detectable LOX2 expression. Lineage tracer injections showed that the MPS neurons arise from a stereotyped cell lineage and are descended from the O teloblast stem cell. In midbody ganglia 2 to 6 and 18 to 21, there are lineally homologous neurons that do not express either LOX2 protein or SCP. Thus, the boundaries of LOX2 expression coincide precisely with two of the segmental boundaries of MPS differentiation, suggesting that expression of LOX2 at the level of this single identified neuron governs some, but not all, aspects of the neuron's segmental diversification.

Animals↗
PubMed1996

Kainate responses of leech Retzius neurons in situ and in vitro.

Responses to the ionotropic glutamate receptor agonist kainate were measured in Retzius cells (RCs) of intact segmental ganglia (in situ), acutely isolated RCs, and cultured RCs (in vitro) of the leech Hirudo medicinalis. RCs in intact ganglia responded to kainate (5-20 microM) with depolarizations up to 30 mV or with an inward current under voltage-clamp that reversed near -10 mV. The membrane conductance increased by a factor of 2.5 at a holding potential of -70 mV in the presence of 20 microM kainate. In RCs in situ the membrane responses to 5 microM kainate increased when applied repeatedly 3-5 times. After this potentiation, the amplitude and time course of the membrane responses to 5 microM kainate were similar to the membrane response to 20 microM kainate. In current-clamp experiments kainate evoked an increase in intracellular calcium concentrations ([Ca2+]i) only when the membrane depolarized beyond -40 mV. In voltage-clamped RCs at a holding potential of -70 mV, kainate caused no significant rise in [Ca2+]i, indicating that the Ca2+ permeability of these kainate-gated ion channels appears to be negligible. The potentiation of the kainate-induced responses in RCs in situ was also present in voltage-clamped cells, where no or only small changes in [Ca2+]i occurred, suggesting that the underlying mechanism seemed to be independent of intracellular Ca2+ changes. In addition, the potentiation of the kainate-induced membrane responses was unaffected by cyclothiazide (100 microM), concanavalin A (0.5 mg/mL), and in the presence of extracellular low-Ca2+ and high-Mg2+ concentrations to suppress synaptic transmission in the ganglion. During whole-cell patch-clamp recordings (up to 50 min) potentiation remained the same indicating that small intracellular messenger molecules, which would be expected to dissipate, were not likely to be involved in mediating this potentiation. In acutely isolated RCs kainate induced no or only very small voltage responses. A potentiation of the kainate response was never observed in acutely isolated RCs. In cultured RCs (2-7 days in vitro) kainate evoked membrane responses with no apparent potentiation. Cultured RCs also responded with Ca2+ transients only when depolarized beyond -40 mV. The results show that RCs respond differently to kainate when kept isolated in culture compared to RCs in intact ganglia. The mechanism underlying the potentiation of the kainate response of RCs in situ, however, could not yet be identified.

Animals↗